The visual examination of biological material plays a central role in the detection of pathological alterations. In the field of dermato-surgery it is e.g. routinely tested whether the cutting margins of tissue material which has been surgically removed are located in sano, i.e. in the healthy tissue, to make sure that the tumor material has been completely removed. It has been demonstrated that this histological control of the three-dimensional margins of excisions, which is also referred to as three-dimensional histology, reduces the risk of local recurrences and is, therefore, the method of first choice for many tumors of the skin.
So far, the visual examination of the cutting margins within the context of the three-dimensional histology is realized at tissue sections which were previously prepared from the removed biological material.
In the USA and in parts also in Europe the control of the cutting margins is made with frozen or quick sections. This method is also referred to as “Mohs' Surgery” according to its originator Frederic E. Mohs. In this method the removed biological material is deep frozen. Out of this frozen tissue sections of a thickness of about 5 to 10 μm are prepared. The sections are then visually examined by transmission microscopy. The results of this examination are available within 30 to 45 minutes. However the frozen or quick section methods are imprecise and can only be used in a reliable manner for solid, nodular basal cell carcinomas.
More precise results can be obtained by the so-called paraffin section method. In this method the removed biological material is embedded into paraffin. Then tissue sections are prepared and again examined by transmission microscopy. However, because of the complex sample preparation the results of the examination are only available about 20 hours after the removal of the biological material. For this reason it is e.g. not possible, in case of the detection of branches of a tumor at the cutting margin, to make a subsequent excision in a targeted manner within the same surgery. As a consequence, the cutting areas or wounds are often kept open until the result is available which means a high stress and high risk of infection for the patient.
Another decisive disadvantage of the before-mentioned section methods is the fact that the examined biological material is so to say “consumed” and no longer available for e.g. histological or molecular biological examinations and other processes. If such further examinations would be necessary biological material is again to be taken from the affected patient which could again require narcoses and other surgical risks.
The confocal laser scanning microscopy is a method where so-called optical sections are prepared allowing a histopathological assessment of biological material. The depth of penetration is about 0.3 mm allowing the evaluation of the tumor excision from the cutting margin from the outside. However the confocal laser scanning microscopy is technically complex and the diagnostic reliability is insufficient.